Journal: Cancer Chemotherapy and Pharmacology

Article Title: Preclinical pharmacokinetics, pharmacodynamics, and efficacy of RG7116: a novel humanized, glycoengineered anti-HER3 antibody

doi: 10.1007/s00280-015-2697-8

Figure Lengend Snippet: a Dose–response study (FaDu_001) investigating the efficacy, pharmacodynamics, and pharmacokinetics of three-weekly ip doses of RG7116 (0.3–10 mg/kg) in female SCID-beige mice bearing subcutaneous human hypopharyngeal cancer FaDu cells. b Dose-dependent tumor growth inhibition was observed in mice treated with three doses of weekly RG7116. c Expression of HER3 and pHER3 was down-modulated in mice receiving RG7116 at doses above 1 mg/kg. d Accumulation of RG7116 was seen with doses above 1 mg/kg but not with the 0.3 mg/kg dose. IHC immunohistochemistry, SEM standard error of the mean, SD standard deviation

Article Snippet: Tissue was formalin-fixed and paraffin-embedded, and standard immunohistochemistry (IHC) was conducted using a murine mAb specific for HER3 (clone DAK-H3-IC, DAKO, #M7297) and rabbit mAb specific for phosphorylated HER3 (pHER3; clone 21D3, Cell Signaling Technologies, #4791).

Techniques: Drug discovery, Inhibition, Expressing, Immunohistochemistry, Standard Deviation

Journal: Cancer Chemotherapy and Pharmacology

Article Title: Preclinical pharmacokinetics, pharmacodynamics, and efficacy of RG7116: a novel humanized, glycoengineered anti-HER3 antibody

doi: 10.1007/s00280-015-2697-8

Figure Lengend Snippet: a Pharmacokinetics and pharmacodynamics of RG7116 following a single i.v. administration of an efficacious (1 mg/kg) and less-efficacious (0.3 mg/kg) dose of RG7116 in FaDu xenograft mice (FaDu_006). b PK profiles for the two doses were similar until approximately 87 h post-dose, after which rapid elimination of antibody was seen with the lower dose. c Inhibition of pHER3 and pAKT ( d ) was seen with both doses initially; however, this was more prolonged with the efficacious dose. e An inverse correlation was seen between RG7116 exposure and both pHER3 and pAKT inhibition, with faster elimination in the 0.3 mg/kg group consistent with an earlier return to baseline pHER3/HER3 levels. SD standard deviation

Article Snippet: Tissue was formalin-fixed and paraffin-embedded, and standard immunohistochemistry (IHC) was conducted using a murine mAb specific for HER3 (clone DAK-H3-IC, DAKO, #M7297) and rabbit mAb specific for phosphorylated HER3 (pHER3; clone 21D3, Cell Signaling Technologies, #4791).

Techniques: Drug discovery, Inhibition, Standard Deviation

Journal: Cancer Chemotherapy and Pharmacology

Article Title: Preclinical pharmacokinetics, pharmacodynamics, and efficacy of RG7116: a novel humanized, glycoengineered anti-HER3 antibody

doi: 10.1007/s00280-015-2697-8

Figure Lengend Snippet: a Extended dosing study investigating seven i.v. weekly administrations of an efficacious dose (3 mg/kg) and a less-efficacious (0.3 mg/kg) dose of RG7116 in FaDu xenograft mice (FaDu_008). RG7116 given as a three-weekly cycle (two doses in total) was also investigated. b Dose-dependent efficacy up to Day 39 was seen as in the previous study and tumor growth inhibition with 3 mg/kg RG7116 given as a three-weekly cycle was similar to weekly dosing. Mice in the 0.3 mg/kg group were rechallenged ( black arrow ) with 5 mg/kg on Day 39 resulting in tumor growth inhibition for two further assessments; however, tumor regrowth was observed in all dosing groups from Day 50. c RG7116 trough concentrations from the first to third administrations again showed accumulation only with the higher dose. Inhibition of HER3 ( d ) and AKT ( e ) phosphorylation was also seen with weekly dosing at the efficacious dose (3 mg/kg) but not with the lower dose (0.3 mg/kg). Following a single 3 mg/kg dose (representing a three-weekly schedule), initial inhibition of HER3 phosphorylation was seen to diminish by Day 14 and 21. f Upregulation of HER1 (~2.1-fold) and HER2 (~1.3-fold) mRNA was seen 96 h after FaDu xenograft mice ( n = 5) were administered 1.0 mg/kg RG7116 in a separate study, and this was associated with upregulation of HER1 and HER2 protein. g To compare the single animals, 20 µg total protein lysate was loaded per lane (lanes 101–110 are vehicle and 201–210 are the RG7116-treated animals). At Day 52, inhibition of HER3 phosphorylation was maintained in RG7116-treated mice, and expressed HER1 was seen to be phosphorylated compared to vehicle control. HER2 and pHER2 were not changed (data not shown). SEM standard error of the mean, SD standard deviation

Article Snippet: Tissue was formalin-fixed and paraffin-embedded, and standard immunohistochemistry (IHC) was conducted using a murine mAb specific for HER3 (clone DAK-H3-IC, DAKO, #M7297) and rabbit mAb specific for phosphorylated HER3 (pHER3; clone 21D3, Cell Signaling Technologies, #4791).

Techniques: Inhibition, Phospho-proteomics, Control, Standard Deviation

Journal: Cancer Chemotherapy and Pharmacology

Article Title: Preclinical pharmacokinetics, pharmacodynamics, and efficacy of RG7116: a novel humanized, glycoengineered anti-HER3 antibody

doi: 10.1007/s00280-015-2697-8

Figure Lengend Snippet: Down-modulation of HER3 and inhibition of HER3 phosphorylation in surrogate tissues. a Compared to baseline, levels of HER3 were markedly diminished in oral mucosa and skin biopsies at 2 and 6 h post-treatment in a cynomolgus monkey treated with 20 mg/kg RG7116. b IHC shows expression of pHER3 in skin tissue and was also reduced following treatment. (IHC was scored from 0 to +++; + to ++ indicates positive staining and 0 to + indicates borderline/negative staining)

Article Snippet: Tissue was formalin-fixed and paraffin-embedded, and standard immunohistochemistry (IHC) was conducted using a murine mAb specific for HER3 (clone DAK-H3-IC, DAKO, #M7297) and rabbit mAb specific for phosphorylated HER3 (pHER3; clone 21D3, Cell Signaling Technologies, #4791).

Techniques: Inhibition, Phospho-proteomics, Expressing, Staining, Negative Staining

Journal: Cell Death & Disease

Article Title: miR-148a-3p represses proliferation and EMT by establishing regulatory circuits between ERBB3/AKT2/c-myc and DNMT1 in bladder cancer

doi: 10.1038/cddis.2016.373

Figure Lengend Snippet: Clinical characteristics of the bladder cancer patients

Article Snippet: The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-AKT2, anti-p-AKT, anti-Rb, anti-p-Rb (Epitomics, Burlingame, CA, USA), anti-E2F1, anti-CDK4 and anti-DNMT1 (Proteintech, Chicago, IL, USA), anti-N-cadherin, anti-p27, anti-p21, anti-c-myc, anti-ERBB3, and anti-CCND1 (Cell Signaling Technology, Beverly, MA, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: miR-148a-3p represses proliferation and EMT by establishing regulatory circuits between ERBB3/AKT2/c-myc and DNMT1 in bladder cancer

doi: 10.1038/cddis.2016.373

Figure Lengend Snippet: ERBB3 and AKT2 are direct miR-148a-3p targets. ( a ) The miR-148a-3p- targeting sites in ERBB3 and AKT2 3′-UTRs were mutated. ( b ) Decreased ERBB3 and AKT2 expression was observed in miR-148a-3p-transfected T24 and UM-UC-3 cells via qRT-PCR and western blot. ( c ) miR-148a-3p significantly suppressed the luciferase activity of vector that carried the ERBB3 and AKT2 3′-UTRs but not control vector. ( d ) Statistical analysis indicated that ERBB3 expression was significantly higher in bladder cancer tissues than in adjacent non-tumor tissues. ( e ) Representative images of IHC staining of TMA. ERBB3 localized to the membrane. ( f ) miR-148a-3p was significantly negatively correlated with ERBB3 ( r =0.149, P =0.017) and DNMT1 ( r =0.108, P =0.041). ERBB3 was significantly negatively correlated with DNMT1 ( r =0.116, P =0.042). Error bars represent the S.E. obtained from three independent experiments; * P <0.05. Scale bar=100 μ m

Article Snippet: The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-AKT2, anti-p-AKT, anti-Rb, anti-p-Rb (Epitomics, Burlingame, CA, USA), anti-E2F1, anti-CDK4 and anti-DNMT1 (Proteintech, Chicago, IL, USA), anti-N-cadherin, anti-p27, anti-p21, anti-c-myc, anti-ERBB3, and anti-CCND1 (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Plasmid Preparation, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: miR-148a-3p represses proliferation and EMT by establishing regulatory circuits between ERBB3/AKT2/c-myc and DNMT1 in bladder cancer

doi: 10.1038/cddis.2016.373

Figure Lengend Snippet: Univariate and multivariate analyses of predictors of overall survival in bladder cancer patients

Article Snippet: The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-AKT2, anti-p-AKT, anti-Rb, anti-p-Rb (Epitomics, Burlingame, CA, USA), anti-E2F1, anti-CDK4 and anti-DNMT1 (Proteintech, Chicago, IL, USA), anti-N-cadherin, anti-p27, anti-p21, anti-c-myc, anti-ERBB3, and anti-CCND1 (Cell Signaling Technology, Beverly, MA, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: miR-148a-3p represses proliferation and EMT by establishing regulatory circuits between ERBB3/AKT2/c-myc and DNMT1 in bladder cancer

doi: 10.1038/cddis.2016.373

Figure Lengend Snippet: ERBB3 knockdown suppresses bladder cancer cell proliferation. ( a ) Three different and effective siRNAs were used in our studies to avoid off-target phenomena. ( b ) CCK-8 assay. The relative cell viability of the Si-ERBB3 of T24 and UM-UC-3 cells was lower than that of NC-treated groups (cell viability of 0 nM was regarded as 1.0). ( c ) Colony formation assay (representative wells are presented). The colony formation rate was lower for Si-ERBB3 (50 nM)-transfected groups compared with NC (50 nM)-transfected groups. ( d ) Flow cytometric analysis (representative images are presented) of cell cycle distribution. ERBB3 knockdown induced a significant accumulation of cells in the G1-phase and blocked entry into G1-S. ( e ) Western blot analysis. Si-ERBB3 (50 nM) inhibited the cell cycle and AKT2 signaling-related proteins in T24 and UM-UC-3 cells. Error bars represent the S.E. obtained from three independent experiments; * P <0.05

Article Snippet: The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-AKT2, anti-p-AKT, anti-Rb, anti-p-Rb (Epitomics, Burlingame, CA, USA), anti-E2F1, anti-CDK4 and anti-DNMT1 (Proteintech, Chicago, IL, USA), anti-N-cadherin, anti-p27, anti-p21, anti-c-myc, anti-ERBB3, and anti-CCND1 (Cell Signaling Technology, Beverly, MA, USA).

Techniques: CCK-8 Assay, Colony Assay, Transfection, Western Blot

Journal: Cell Death & Disease

Article Title: miR-148a-3p represses proliferation and EMT by establishing regulatory circuits between ERBB3/AKT2/c-myc and DNMT1 in bladder cancer

doi: 10.1038/cddis.2016.373

Figure Lengend Snippet: ERBB3 knockdown suppresses bladder cancer cell motility. ( a ) Transwell assay (representative micrographs are presented). Si-ERBB3 (50 nM) impaired T24 and UM-UC-3 cells motility. ( b ) Western blot analysis. Si-ERBB3 (50 nM) inhibited EMT-related protein expression in T24 and UM-UC-3 cells. Error bars represent the S.E. obtained from three independent experiments; * P <0.05. Scale bar=100 μ m

Article Snippet: The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-AKT2, anti-p-AKT, anti-Rb, anti-p-Rb (Epitomics, Burlingame, CA, USA), anti-E2F1, anti-CDK4 and anti-DNMT1 (Proteintech, Chicago, IL, USA), anti-N-cadherin, anti-p27, anti-p21, anti-c-myc, anti-ERBB3, and anti-CCND1 (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Transwell Assay, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: miR-148a-3p represses proliferation and EMT by establishing regulatory circuits between ERBB3/AKT2/c-myc and DNMT1 in bladder cancer

doi: 10.1038/cddis.2016.373

Figure Lengend Snippet: Inhibition of miR-148a-3p expression partially rescues Si-ERBB3-induced suppression of cell proliferation and EMT. ( a and b ) Co-transfection of miR-148a-3p-Inh attenuated the ERBB3 expression inhibited by Si-ERBB3 at the mRNA and protein level in both bladder cancer cell lines. Similar p-AKT protein expression pattern was also observed. ( c – e ) Transfection of miR-148a-3p-Inh partially but significantly rescued the Si-ERBB3-induced inhibition of cell proliferation, cell cycle and motility in T24 cell line. Error bars represent the S.E. obtained from three independent experiments; * P <0.05. Scale bar=100 μ m

Article Snippet: The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-AKT2, anti-p-AKT, anti-Rb, anti-p-Rb (Epitomics, Burlingame, CA, USA), anti-E2F1, anti-CDK4 and anti-DNMT1 (Proteintech, Chicago, IL, USA), anti-N-cadherin, anti-p27, anti-p21, anti-c-myc, anti-ERBB3, and anti-CCND1 (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Inhibition, Expressing, Cotransfection, Transfection

Journal: Cell Death & Disease

Article Title: miR-148a-3p represses proliferation and EMT by establishing regulatory circuits between ERBB3/AKT2/c-myc and DNMT1 in bladder cancer

doi: 10.1038/cddis.2016.373

Figure Lengend Snippet: miR-148a-3p/ERBB3/AKT2/c-myc established a regulatory circuit in bladder cancer regulation. ( a ) Transfection of Si-ERBB3 decreased the expression level of its downstream gene AKT2 and c-myc by qRT-PCR. ( b ) Transfection of Si-AKT2 decreased the phosphorylation level of AKT and expression of c-myc. ( c ) Overexpression of c-myc was able to, at least partially, rescue the level of c-myc in the presence of Si-AKT2. ( d ) Both AKT-Inh and c-myc-Inh decreased the expression of c-myc on both mRNA and protein levels. ( e ) qRT-PCR. c-myc overexpression decreased miR-148a-3p expression. ( f ) c-myc overexpression in HEK293T cells increased the luciferase activity of the miR-148a-3p promoter. Co-treatment with c-myc-Inh diminished the increase in luciferase activity. ( g ) c-myc-binding region in the miR-148a-3p promoter region. Error bars represent the S.E. obtained from three independent experiments; * P <0.05

Article Snippet: The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-AKT2, anti-p-AKT, anti-Rb, anti-p-Rb (Epitomics, Burlingame, CA, USA), anti-E2F1, anti-CDK4 and anti-DNMT1 (Proteintech, Chicago, IL, USA), anti-N-cadherin, anti-p27, anti-p21, anti-c-myc, anti-ERBB3, and anti-CCND1 (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Over Expression, Luciferase, Activity Assay, Binding Assay

Journal: Cell Death & Disease

Article Title: miR-148a-3p represses proliferation and EMT by establishing regulatory circuits between ERBB3/AKT2/c-myc and DNMT1 in bladder cancer

doi: 10.1038/cddis.2016.373

Figure Lengend Snippet: Schematic diagram showing that miR-148a-3p/ERBB3/AKT2/c-myc and DNMT1 established a bi-loop to regulate bladder cancer

Article Snippet: The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-AKT2, anti-p-AKT, anti-Rb, anti-p-Rb (Epitomics, Burlingame, CA, USA), anti-E2F1, anti-CDK4 and anti-DNMT1 (Proteintech, Chicago, IL, USA), anti-N-cadherin, anti-p27, anti-p21, anti-c-myc, anti-ERBB3, and anti-CCND1 (Cell Signaling Technology, Beverly, MA, USA).

Techniques: